Vaccines for nontypable haemophilus influenzae

ABSTRACT

Protein &#34;e&#34; of H. influenzae, a lipoprotein of approximately 28,000 daltons, has been purified and sequenced. Protein &#34;e&#34; and peptides or proteins having a shared epitope, can be used to vaccinate against nontypable (and typable) H. influenzae and to prevent otitis media caused by H. influenzae. For this purpose, protein &#34;e&#34; or derivatives thereof can be produced in native, synthetic or recombinant forms and can be administered alone or in conjunction with other antigens of H. influenzae. Protein &#34;e&#34; can also be used in multivalent vaccines designed for H. influenzae and one or more other infectious organisms.

RELATED APPLICATION

This is a divisional of copending application Ser. No. 07/491,466, filedon Mar. 9, 1990, now U.S. Pat. No. 5,601,831, issued on Feb. 11, 1993,which is a continuation-in-part of Ser. No. 07/320,971, filed Mar. 9,1989, now abandoned. The teachings are incorporated by reference herein.

BACKGROUND

Haemophilus influenzae are divided into two groups of strains, typableand nontypable. Strains which possess a known capsule are typed by theserological reaction of the capsule with reference antisera. Types a-fhave been identified. Strains which fail to react with any of thereference antisera are nontypable.

H. influenzae type b (Hib) is the most frequent cause of neonatalmeningitis and other invasive infections in the United States (Fraser etal., 1974, Am. J. Epidemiol. 100:29-34). The major incidence ofchildhood meningitis occurs between the ages of one and five years.Sixty percent of the meningitis cases due to Hib occur in children underthe age of two years (Fraser et al., supra).

It is now well established that nontypable H. influenzae also causediseases including pneumonia, bacteremia, meningitis, postpartum sepsis,and acute febrile tracheobronchitis in adults (Murphy et al., 1985, J.Infect. Diseases 152:1300-1307). In addition, nontypable H. influenzaeare a frequent etiologic agent of otitis media in children and youngadults. Indeed, about 20 to 40% of all cases of otitis media can beattributed to H. influenzae. Children may experience multiple infectionsof the same organism since infection confers no long lasting immunity.Currently, chronic or repeat otitis media is treated by administrationof antibiotics and, if necessary, by drainage of the inner ear. H.influenzae strains have also been implicated as a primary cause ofsinusitis (Cherry J. D. and J. P. Dudley, 1981, in Textbook of PediatricInfectious Diseases, Feigin and Cherry eds., pp 103-105). Additionally,nontypable H. influenzae cause neonatal sepsis.

Antiserum produced against the capsular polysaccharide of Hib,polyribosyl ribitol phosphate (PRP), has been shown to be bactericidaland protective against Hib (Smith et al., 1973, Pediatrics 52:637-644;Anderson, et al., 1972, J. Clin. Inv. 51:31-88). Anti-PRP antibody,however, is ineffective against nontypable H. influenzae infection.

Currently available vaccines against H. influenzae are all directedagainst Hib. All are effective by eliciting anti-PRP antibody. Anti-PRPantibody, however, is ineffective against nontypable H. influenzae,which by definition lack the PRP capsule. There is a long recognizedneed for a vaccine that will protect against nontypable H. influenzae.

SUMMARY OF THE INVENTION

This invention pertains to the outer membrane protein "e" of H.influenzae and to peptides and proteins which have an epitope in commonwith protein "e". Protein "e" is a lipoprotein which has a molecularweight of about 28,000 daltons and an amino acid sequence as set forthin FIG. 7. The invention also pertains to the use of protein "e" andpeptides and proteins having protein "e" epitopes for vaccinationagainst nontypable and typable H. influenzae. The peptides and proteinscan be used in univalent vaccines or in multivalent vaccines inconjunction with other antigens of typable or nontypable H. influenzae(e.g., as mixtures, fusion or conjugates therewith) or with antigens ofother infectious bacteria, viruses or parasites. The peptides andproteins elicit biologically active (bactericidal and/or opsonic)antibody against H. influenzae. Importantly, protein "e" acts in synergywith other outer membrane proteins of H. influenzae in elicitingcrossreactive, bactericidal antibody responses, especially againstnontypable strains of H. influenzae, and thus, the peptides or proteinsof this invention are particularly effective when administered togetherwith these proteins. In addition, antibody specific for epitopes ofprotein "e" can be used (either alone or in conjunction with antibodyagainst epitopes of other outer membrane proteins) for passiveimmunization against H. influenzae and in diagnostic assays for theorganism.

The invention also pertains to methods of producing native, purifiedprotein "e", and to various vaccine formulations containing them.Protein "e" can be obtained by purification from H. influenzae. Thisinvention also provides a method of isolating and purifying protein "e"in native lipoprotein form from H. influenzae by differential detergentextraction to provide an essentially endotoxin-free preparation withoutthe use of agents considered harmful to humans. Protein "e" can also beproduced by recombinant DNA techniques in lipidated or nonlipidated formor by protein synthesis. Epitopic oligopeptides and other fragments ofprotein "e" and analogues of these can be produced by recombinant DNAtechniques, chemical synthesis or chemical or enzymatic cleavage. Thesepeptides or proteins, in turn, can be fused or conjugated to otherantigens of H. influenzae or to antigens of other microorganisms(bacteria, viruses, fungi or parasites) by chemical or genetic couplingtechniques to produce multivalent antigenic conjugates and fusionpeptides or proteins. The peptides or proteins can be modified forconjugation such as by addition of amino acids or other coupling groups.For vaccination, the peptides or proteins, in any of the formsdescribed, can be formulated in pharmaceutically acceptable vehicleswith optional additives such as adjuvants.

The invention also pertains to isolated nucleic acid sequences whichencode the native protein "e" or any of the various peptidic orproteinaceous derivatives of the protein "e". The sequences can beincorporated into appropriate expression systems for production ofprotein "e" or any of the derived peptides and proteins of thisinvention. In addition, the gene fragments or oligonucleotides can beused as probes in nucleic acid hybridization assays.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the analysis of purified protein "e" by sodium dodecylsulfate polyacrylamide gel electrophoresis.

FIG. 2 (a and b) shows the results of an immunoblot analysis of thereactivity of antibodies against protein "e" with isolates of nontypableH. influenzae.

FIG. 3 shows reactivity of anti-e monoclonal antibodies vs. E. coliHB101(pPX504).

FIG. 4 is a map of plasmid pPX513.

FIG. 5 shows reactivity of anti-e monoclonal antibodies vs.HB101(pPX513).

FIG. 6 shows DNA sequence of the coding region of the protein "e" genefrom H. influenzae and derived amino acid sequence.

FIG. 7 shows amino acid sequence of the protein "e". The amino acidsequence of the mature protein "e" shown above is derived from the DNAsequence. The underlined sequence has been confirmed by amino acidsequencing of the peptides obtained from digestion of the purified "e"protein with several endoproteinases.

FIG. 8 shows the hybridization of pPX504 to Haemophilus chromosomal DNA.

DETAILED DESCRIPTION OF THE INVENTION

Protein "e" is an outer membrane protein of H. influenzae which has amolecular weight of about 28,000 daltons and an amino acid sequence asshown in FIG. 7. It has now been found that protein "e" exists as alipoprotein in association with the outer membrane-cell wall complex ofthe bacteria.

Protein "e" has several properties which make it (and peptides andproteins having epitopes of protein "e") especially valuable forvaccination against nontypable H. influenzae. Protein "e" is capable ofeliciting a bactericidal immune response against nontypable H.influenzae. Importantly, protein "e" is highly conserved among H.influenzae strains. The protein has been detected both by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western Blotanalysis in all H. influenzae strains tested, and in addition,monoclonal antibody data indicate that the protein is highly conserved.Thus, the protein can induce an immune response against differentstrains of nontypable H. influenzae. Further, protein "e" elicitsbactericidal antibodies which act in synergy with antibodies againstother outer membrane proteins of H. influenzae. Because of this, theprotein can be used in conjunction with other outer membrane proteins toinduce a more potent bactericidal response.

This invention encompasses substantially pure protein "e" and peptidesand proteins having epitopes of protein "e". The peptides or proteinsbear a common epitope with protein "e" (and thus are immunologicallycrossreactive therewith). They can include fragments or oligopeptidescontaining epitopes of protein "e" as described below. The amino acidsequence of protein "e" has been determined and is shown in FIG. 7. Thepeptides and proteins of this invention comprise any peptide or proteinhaving at least a portion of the amino acid sequence depicted in FIG. 7or any biologically equivalent sequences. Altered sequences includesequences in which functionally equivalent amino acid residues aresubstituted for residues within the sequence resulting in a silentchange. For example, one or more amino acid residues within the sequencecan be substituted by another amino acid of a similar polarity whichacts as a functional equivalent, resulting in a silent alteration.Substitutes for an amino acid within the sequence may be selected fromother members of the class to which the amino acid belongs. For example,the nonpolar (hydrophobic) amino acids include glycine, alanine,leucine, isoleucine, valine, proline, phenylalanine, tryptophan andmethionine. The polar neutral amino acids include serine, threonine,cysteine, tyrosine, asparagine, and glutamine. The charged (basic) aminoacids include arginine, lysine and histidine. The negatively charged(acidic) amino acids include aspartic and glutamic acid.

The peptides and proteins of this invention also include fragments oroligopeptides having epitopes of protein "e" represented within thesequence or any analogues of such fragments or epitopes. In addition,any of the peptides and proteins can be modified for conjugation toother molecules, e.g. by the attachment of coupling groups such as theamino acids cysteine and lysine or other linking groups.

As described in detail below, protein "e" and the peptides and proteinsof this invention can be used in many different forms, (e.g. alone, inmixtures or as conjugates and fusions) in vaccines and in diagnosticmethods. For these purposes, the peptides and proteins can be producedby isolation from H. influenzae, by chemical synthesis, or by expressionas recombinant molecules. The methods of using the peptides and proteinsof this invention and the techniques for their production are discussedbelow.

Purification of Protein "e"

Native protein "e" can be purified from H. influenzae by a procedure ofdifferential detergent extraction. The procedure is based on the use ofsulfobetaine detergents which can selectively extract outer membraneproteins of H. influenzae. The procedure does not involve the use ofdenaturants and reducing agents such as sodium dodecylsulfate and2-mercaptoethanol, respectively, (see Munson et al., 1984, Infect.Immun. 49:544-49) which can destroy important antigenic epitopes of theprotein and which are not widely accepted as safe for administration tohumans.

The procedure entails first obtaining outer membrane components of H.influenzae cells. Outer membrane components can be prepared from a totalcell membrane fraction. Total membrane fractions are typically preparedby differential sedimentation after disruption of H. influenzae cells bymethods such as sonication, grinding, or expulsion from a french pressor other homogenization device. The total membrane fraction is thenfractionated into inner and outer membranes by density gradientsedimentation or by differential solubilization of the inner membraneconstituents with certain detergents such as polyoxyethyleneoctylphenol(Triton X-100™) or N-lauroyl sarcosine, sodium salt (sarcosyl). In thepreferred embodiment, outer cell membrane components are prepared bydifferential solubilization of inner membranes in 0.1-2% (w/v) TritonX-100™ in 10 mM HEPES-NaOH 1 mM MgCl₂, pH 7.4. This extraction istypically performed twice.

As an alternate source of outer membrane components, a culture medium ofH. influenza cells can be used. The medium contains shed components(called "blebs") of the outer membrane of the bacteria. See Loeb, M. R.(1987) Infection and Immunity 55(11):2612-2618.

A subfraction of the preparation of outer cell membrane components whichis enriched in protein "e" can be produced by extraction with an aqueoussolution of 0.1-2.0% (preferably 1%) sarcosyl at pH 8.0. This extractionis typically performed two or three times and it removes a major proteincomponent as well as other materials.

Solubilization of the protein "e" from the outer membrane-cell wallcomplex can then be achieved by a two-step differential solubilizationwith sulfobetaine detergents. In the first step, an aqueous solution of0.1-10%, typically 0.1-2% (w/v) dodecylsulfobetaine (Zwittergent™ 3-12)is used to remove outer membrane proteins other than protein "e".Preferably, a 1% solution is used and the extraction is usuallyperformed 2-3 times. The residual insoluble components are thenextracted with an aqueous solution of tetradecyl- orhexadecylsulfobetaine (Zwittergent™ 3-14 or 3-16) under the sameconditions. This extraction results in the solubilization of protein"e".

After solubilization, further purification of protein "e" can beachieved by standard methods including ion exchange, molecular sieve,hydrophobic, reverse phase or adsorption (e.g. hydroxylapatite)chromatography, affinity chromatography, chromatofocusing, isoelectricfocusing and preparative electrophoresis.

Protein "e" purified by this method is substantially free of bacterialendotoxin and is suitable for administration to humans. The purifiedpreparation of protein "e" can be formulated alone as a vaccine for H.influenzae or in a mixture with antigens of other organisms implicatedin otitis media. If desired, the protein can be fragmented by standardchemical or enzymatic techniques to produce antigenic segments.

Preparation of the peptides and proteins by chemical synthesis

The peptides and proteins of this invention can be chemicallysynthesized according to the amino acid sequence shown in FIG. 7 orvariations of this sequence as described above. Any of the standardchemistries for solid or liquid phase synthesis of peptides and proteinsmay be used. Chemical synthesis may be particularly suitable forproduction of oligopeptides containing epitopes of protein "e".

Preparation of the peptides and proteins by recombinant DNA techniques

Protein "e" and the peptides and proteins which share epitopes ofprotein "e" can be produced by recombinant DNA techniques. In general,these entail obtaining by synthesis or isolation a DNA sequence whichencodes the derived peptide or protein and introducing it into anappropriate vector/host expression system where it is expressed. The DNAcan consist of the gene encoding protein "e" or any segment of the genewhich encodes a useful segment of the protein "e". The DNA can be fusedto DNA encoding other antigens of H. influenzae or antigens of otherbacteria, viruses, parasites or fungi to create genetically fused(sharing a common peptide backbone) multivalent antigens. For example,protein "e" can be fused to other outer membrane proteins (or fragmentsor epitopes thereof) of H. influenzae to yield fusion proteinscomprising multiple outer membrane protein determinants.

Genetic engineering techniques can also be used to characterize, modifyand/or adapt the encoded peptides or proteins. For example, sitedirected mutagenesis of the gene encoding protein "e" can be used toidentify regions of the protein responsible for generation of protectiveantibody responses (e.g., bactericidal or opsonic epitopes). Thesetechniques can also be used to modify the protein in regions outside theprotective domains, for example, to increase the solubility of theprotein to allow easier purification.

Obtaining DNA encoding protein "e"

DNA encoding protein "e" or fragments thereof, can be synthesizedchemically according to the nucleotide sequence shown in FIG. 6. Severaltechniques are available for synthesizing DNA of desired nucleotidesequences. See, e.g., Matteucci et al., J. Am. Chem. Soc. (1981)103:3185; Alvarado-Urbina et al., Science (1980) 214:270. A preferredtechnique for synthesis of DNA segments is the β-cyanoethylphophoramidite chemistry. See e.g., Sinha, N. D. et al., Nucleic AcidsResearch 13:4539 (1984). The synthesized DNA can be adapted forinsertion into appropriate vectors by techniques described below forisolated DNA.

As an alternative to chemical synthesis, DNA encoding protein "e" can beisolated from Haemophilus influenzae. Any H. influenzae strain can serveas the source for the protein "e" gene. Since many H. influenzae strainscontain no detectable plasmids or inducible prophages, the protein "e"gene is probably chromosomal, thus, the gene must be isolated from H.influenzae chromosomal DNA. In the remainder of this section, DNAencoding an H. influenzae gene will be referred to as "Hi DNA", and DNAencoding protein "e" sequences will be referred to as "protein "e" DNA".

In order to generate Hi DNA fragments, the Hi DNA can be cleaved atspecific sites with various restriction enzymes. Alternatively, one mayuse low concentrations of DNase I to fragment the DNA, or the DNA can bephysically sheared, for example, by sonication. The linear DNA fragmentscan then be separated according to size by standard techniques such asagarose and polyacrylamide gel electrophoresis, column chromatography(e.g., molecular sieve or ion exchange chromatography) or velocitysedimentation in sucrose gradients.

Any restriction enzyme or combination of restriction enzymes may be usedto generate the Hi DNA fragment(s) containing the protein "e" sequenceprovided the enzyme(s) does not destroy a desired property (e.g.,immunopotency) of the protein "e" gene product. For example, an epitopeof a protein can consist of from about 7 to about 14 amino acids. Thus,a protein of the size of the protein "e" may have many discrete epitopesand therefore, many partial protein "e" gene sequences can code for anepitope. Consequently many combinations of restriction enzymes can beused to generate DNA fragments which encode amino acid sequencescorresponding to different antigenic determinants of protein "e".

Once the DNA fragments are generated, identification of the specific DNAfragment containing the protein "e" gene can be accomplished in a numberof ways.

The DNA sequences containing the protein "e" gene can be identified byhybridization with a synthetic oligonucleotide probe. Redundantsynthetic oligonucleotide probes can be constructed based upon the aminoacid sequence of the substantially pure protein "e". These syntheticprobes can be radiolabeled with ³² P-adenosine triphosphate and used toscreen Hi DNA libraries for clones containing protein "e"-specific genesequences (see Anderson et al., 1983, Proc. Nat'l Acad. Sci. USA80:6838-42).

Alternatively, the protein "e" DNA may be identified and isolated afterinsertion into a cloning vector in a "shotgun" approach. A large numberof vector-host systems known in the art may be used. Vector systems maybe either plasmids or modified viruses. Suitable cloning vectorsinclude, but are not limited to the viral vectors such as λ vectorsystem λgtll, λgt λWES.tB, Charon 4, and plasmid vectors such as pBR322,pBR325, pACYC177, pACYC184, pUC8, pUC9, pUC18, pUC19, pLG339, pR290,pK37, pKC101 and other similar systems. The vector system must becompatible with the host cell used. Recombinant molecules can beintroduced into cells via transformation, transfection or infection.

When Hi DNA containing a protein "e" gene or gene fragment is insertedinto a cloning vector and used to transform appropriate host cells manycopies of the protein "e" gene or gene fragment can be generated. Thiscan be accomplished by ligating the Hi DNA fragment into a cloningvector which has complementary cohesive termini. If, however, thecomplementary restriction sites are not present, the ends of the DNAmolecules may be modified. Such modification includes producing bluntends by digesting back single-stranded DNA termini or by filling thesingle-stranded termini so that the ends can be blunt-end-ligated.Alternatively, any sitre desired may be produced by ligating nucleotidesequences (linkers) onto the DNA termini. These ligated linkers maycomprise specific chemically synthesized oligonucleotides encodingrestriction site recognition sequences. For example, according to theDNA modification procedure of Maniatis, (see Maniatis et al., 1982,Molecular Cloning, Cold Spring Harbor Laboratory, pp. 107-114) shearedDNA is treated with a restriction methylase (for example, M. EcoRI) andligated to synthetic DNA linkers which encode a restriction site forthat enzyme. The DNA is then treated with restriction enconucleasd tocleave the terminal linkers (but not the modified internal restrictionsites) and ligated to the appropriate vector arms. In an alternativemethod, the cleaved vector and protein "e" DNA fragment may be modifiedby homopolymeric tailing.

Recombinant protein "e" can be produced as a lipidated or nonlipidatedprotein. For example, by using the intact protein "e" gene, includingits native leader-encoding sequence, a lipidated protein "e" can beproduced in host cells such as E. coli. To produce a nonlipidatedprotein "e", the leader-encoding segment of the protein "e" gene caneither be removed or be replaced by a segment which encodes a leadersequence which does not specify a site for fatty acylation in the hostcell.

Identification of a cloned protein "e" DNA can be accomplished byestablishing a chromosomal gene bank of H. influenzae in a vector systemand screening individual clones for the production of protein "e" orpeptide or protein derived from protein "e" by any of the methodsdescribed herein, including, but not limited to specific reaction withpolyclonal or monoclonal antibodies against protein "e" epitopes.

DNA Expression systems

A variety of host-vector systems can be used to express the peptides andproteins of this invention. Primarily the vector system must becompatible with the host cell used. Host-vector systems include but arenot limited to the following: bacteria transformed with bacteriophageDNA, plasmid DNA or cosmid DNA; microorganisms such as yeast containingyeast vectors; mammalian cell systems infected with virus (e.g.,vaccinia virus, adenovirus, etc.); insect cell systems infected withvirus (e.g., baculovirus). The expression elements of these vectors varyin their strength and specificities. Depending upon the host-vectorsystem utilized, any one of a number of suitable transcription andtranslation elements can be used.

In order to obtain efficient expression of the DNA, a promoter must bepresent in the expression vector. RNA polymerase normally binds to thepromoter and initiates transcription of a gene or a group of linkedgenes and regulatory elements (called an operon). Promoters vary intheir "strength", i.e., their ability to promote transcription. It isdesirable to use strong promoters in order to obtain a high level oftranscription and, hence, a high level of DNA expression. Depending uponthe host cell system any one of a number of suitable promoters can beused. For instance, for E. coli, its bacteriphages or plasmids,promoters such as the lac promoter, trp promoter, recA promoter,ribosomal RNA promoter, and P_(R) or P_(L) promoters of coliphage lambdaand others including but not limited to lacUV5, ompF, bla, lpp and thelike, may be used to direct high levels of transcription of adjacent DNAsegments. Additionally, a hybrid trp-lacUV5 (tac) promoter or other E.coli promoters produced by recombinant DNA or other synthetic DNAtechniques may be used to provide for transcription of the inserted DNA.

Bacterial host cells and expression vectors may be chosen which inhibitthe action of the promoter unless specifically induced. In certainoperons the addition of specific inducers is necessary for efficienttranscription of the inserted DNA; for example, the lac operon isinduced by the addition of lactose or IPTG(isopropylthio-beta-D-galactoside). A variety of other operons, such astrp, pro, etc., are under different controls. The trp operon is inducedwhen tryptophan is absent in the growth media; and the P_(L) promoter oflambda can be induced by an increase in temperature in host cellscontaining a temperature sensitive lambda repressor, e.g., cI857. Inthis way, greater than 95% of the promoter-directed transcription may beinhibited in uninduced cells. Thus, expression of the recombinantpeptide or protein can be controlled. This is important if theexpression product of the DNA is lethal or detrimental to the hostcells. In such cases, transformants may be cultured under conditionssuch that the promoter is not induced; then, when the cells reach asuitable density in the growth medium, the promoter can be induced forproduction of the protein.

One such promoter/operator system is the so-called "tac" or trp-lacpromoter/operator system (Russell and Bennett, 1982, Gene 20:2312;DeBoer, European Patent Application, 67, 540 filed May 18, 1982). Thishybrid promoter is constructed by combining the -35 b.p. (-35 region) ofthe trp promoter and the -10 b.p. (-10 region or Pribnow box) of the lacpromoter (the sequences of DNA which are the RNA polymerase bindingsite). In addition to maintaining the strong promoter characteristics ofthe tryptophan promoter, tac is also controlled by the lac repressor.

When cloning in a eucaryotic host cell, enhancer sequences (e.g., the 72bp tandem repeat of SV40 DNA or the retroviral long terminal repeats orLTRs, etc.) may be inserted to increase transcriptional efficiency.Enhancer sequences are a set of eucaryotic DNA elements that appear toincrease transcriptional efficiency in a manner relatively independentof their position and orientation with respect to a nearby gene. Unlikethe classic promoter elements (e.g., the polymerase binding site and theGoldberg-Hogness "TATA" box) which must be located immediately 5' to thegene, enhancer sequences have a remarkable ability to function upstreamfrom, within, or downstream from eucaryotic genes; therefore, theposition of the enhancer sequence with respect to the inserted DNA isless critical.

Specific initiation signals are also required for efficient genetranscription and translation in procaryotic cells. These transcriptionand translation initiation signals may vary in "strength" as measured bythe quantity of gene specific messenger RNA and protein synthesized,respectively. The DNA expression vector, which contains a promoter, mayalso contain any combination of various "strong" transcription and/ortranslation initiation signals. For instance, efficient translation inE. coli requires a Shine-Dalgarno (SD) sequence about 7-9 bases 5' tothe initiation codon (ATG) to provide a ribosome binding site. Thus, anySD-ATG combination that can be utilized by host cell ribosomes may beemployed. Such combinations include but are not limited to the SD-ATGcombination from the cro gene or the N gene of coliphage lambda, or fromthe E. coli tryptophan E, D. C, B or A genes. Additionally, any SD-ATGcombination produced by recombinant DNA or other techniques involvingincorporation of synthetic nucleotides may be used.

Any of the methods described for the insertion of DNA into an expressionvector can be used to ligate a promoter and other genetic controlelements into specific sites within the vector. H. influenzae sequencesfor expression can be ligated into an expression vector at a specificsite in relation to the vector promoter and control elements so thatwhen the recombinant DNA molecule is introduced into a host cell theforeign genetic sequence can be expressed (i.e., transcribed andtranslated) by the host cell.

The recombinant DNA vector can be introduced into appropriate host cells(bacteria, virus, yeast, mammalian cells or the like) by transformation,transduction or transfection (depending upon the vector/host cellsystem). Host cells containing the vector are selected based upon theexpression of one or more appropriate gene markers normally present inthe vector, such as ampicillin resistance or tetracycline resistance inpBR322, or thymidine kinase activity in eucaryotic host systems.Expression vectors may be derived from cloning vectors, which usuallycontain a marker function. Such cloning vectors may include, but are notlimited to the following: SV40 and adenovirus, vaccinia virus vectors,insect viruses such as baculoviruses, yeast vector, bacteriphage vectorssuch as lambda gt-WES-lambda B, Charon 28, Charon 4A, lambda gt-1-lambdaBC, lambda gt-1-lambda B, M13mp7, M13mp8, M13mp9, or plasmid DNA vectorssuch as pBR322, pAC105, pVA51, pACYC177, pKH47, pACYC184, pUB110, pMB9,pBR325, Col E1, pSC101, pBR313, pML21, RSF2124, pCR1, RP4, pBR328 andthe like.

Transfer of drug resistance factors between H. influenzae and E. colivia conjugation (Stuy, 1979, J. Bact. 139:520-529); and transformation(Mann, 1979, Plasmid 2:503-505) and cloning of Haemophilus chromosomalgenes in E. coli (Deich et al., 1988, J. Bact. 170:489-498; Mann et al.,1980, Gene 3:97-112) indicate that at least some genes can beefficiently expressed in both organisms; and that the basic mechanismsof transcriptional and translational control may be similar.

Expression vectors containing the DNA inserts can be identified by threegeneral approaches: (1) DNA-DNA hybridization using probes comprisingsequences that are homologous to the inserted gene; (2) presence orabsence of "marker" gene functions (e.g., resistance to antibiotics,transformation phenotype, thymidine kinase activity, etc.); and (3)expression of inserted sequences based on the physical immunological orfunctional properties of the gene product.

Once a putative recombinant clone which expresses a protein "e" sequenceis identified, the gene product can be analyzed as follows.Immunological analysis is especially important because the ultimate goalis to use the gene products in vaccine formulations and/or as antigensin diagnostic immunoassays. The peptide or protein should beimmunoreactive. This reactivity may be demonstrated by standardimmunological techniques, such as radio-immunoprecipitation, radioimmunecompetition, ELISA or immunoblots.

Once the gene product is identified as protein "e" or aprotein-e-derived peptide or protein, it can be isolated and purified bystandard methods including chromatography (e.g., ion exchange, affinity,and sizing column chromatography), centrifugation, differentialsolubility, or by any other standard techniques for the purification orproteins. Several techniques exist for purification of heterologousprotein from prokaryotic cells. See e.g., Olson, U.S. Pat. No.4,518,526, Whetzel, U.S. Pat. No. 4,599,197 and Hung et al., U.S. Pat.No. 4,734,362. The purified preparation however produced should besubstantially free of host toxins which might be harmful to humans. Inparticular, when expressed in gram negative bacterial host cells such asE. coli, the purified peptide or protein should be substantially free ofendotoxin contamination.

Evaluating Immunopotency of the peptides and proteins

Experience with antibodies to the capsular polysaccharide of Hib PRP,shows that the ability of the antibodies to kill the bacteria in invitro assays and/or to protect against challenge with Hib in animalmodel systems is closely correlated with the ability to elicit aprotective immune response in human infants.

Anti-protein "e" antibodies elicited in response to the peptides andproteins of this invention can be tested using similar in vitro assaysystems and animal model systems to demonstrate the ability to kill H.influenzae cells and to protect in animal model systems from challengewith H. influenzae. The results from these systems should show a similarcorrelation with the potential of the protein "e" to elicit a protectiveimmune response and to serve in a vaccine for human infants, childrenand adults.

An in vitro complement mediated bactericidal assay system (Musher etal., 1983, Infect. Immun. 39:297-304; Anderson et al., 1972, J. Clin.Invest. 51:31-38) which has been used previously for measuringbactericidal activity of antibodies to PRP and lipopolysaccharide (LPS)against H. influenzae can be used to determine whether or not antibodydirected against a particular peptide protein or fragment thereof hasbactericidal activity against nontypable H. influenzae. These assays canbe performed against a relatively large number of clinical isolates ofnontypable strains to determine whether a broad range of strains arekilled.

Data on the ability of antibody to a particular peptide or protein toprotect against H. influenzae can be obtained using the chinchillaotitis media animal model system. (Barenkamp et al., 1986, Infect.Immun. 52:572-78). In this animal model, chinchillas are challenged byinoculation of the inner ear canal with H. influenzae. An otitis mediamuch like that seen in humans develops. Chinchillas, which have beenimmunized, either actively with outer membrane proteins of H.influenzae, or passively with antibody directed against these proteinsare protected against aural challenge with H. influenzae. (Barenkamp etal., supra). This animal model system could be used to demonstrate theability of antibody to protect against Hi.

Peptides or proteins derived from protein "e" can be evaluated foradditive or synergistic biological activity (e.g. bactericidal and/oropsonic activity). As has been established, protein "e" evokesbactericidal antibodies which act synergistically with antibodiesagainst other outer membrane proteins of H. influenzae. Additive orsynergistic biological activity can be determined by dilutingbactericidal antibodies so that they are no longer bactericidal againstHi and then testing the diluted antibodies in combination with otherantibodies for additive or synergistic activity. Additive or synergisticbiological activity is useful for a combination vaccine composed ofprotein "e" or a fragment or conjugate thereof, and other outer membraneproteins or fragments thereof.

Vaccines

The peptides and proteins of this invention can be used as immunogens insubunit vaccines for vaccination against nontypable H. influenzae. Thevaccines can be used to prevent or reduce susceptibility to acute otitismedia and other diseases caused by nontypable strains of the organism.The vaccines are useful to generally vaccinate children or adultsagainst otitis media or they may be useful for children at risk ofcontracting otitis media (for example, children with a history of earinfection).

The peptides and proteins of this invention can be formulated asunivalent and multivalent vaccines. Protein "e" itself can be used asproduced or isolated by the methods described above. The protein can bemixed, conjugated or fused with other antigens, including B or T cellepitopes of other antigens. In addition to its utility as a primaryimmunogen, protein "e" can be used as a carrier protein to confer orenhance immunogenicity of other antigens.

When a haptenic peptide of protein "e" is used, (i.e., a peptide whichreacts with cognate antibodies, but cannot itself elicit an immuneresponse), it can be conjugated to an immunogenic carrier molecule. Forexample, an oligopeptide containing one or more epitopes of protein "e"may be haptenic. Conjugation to an immunogenic carrier can render theoligopeptide immunogenic. Preferred carrier proteins for the haptenicpeptides of protein "e" are tetanus toxin or toxoid, diphtheria toxin ortoxoid and any mutant forms of these proteins such as CRM₁₉₇. Othersinclude exotoxin A of Pseudomonas, heat labile toxin of E. coli androtaviral particles (including rotavirus and VP6 particles).Alternatively, a fragment or epitope of the carrier protein or otherimmunogenic protein can be used. For example, the happen can be coupledto a T cell epitope of a bacterial toxin. See U.S. patent applicationSer. No. 150,688, filed Feb. 1, 1988, entitled "Synthetic PeptidesRepresenting a T-Cell Epitope as a Carrier Molecule For ConjugateVaccines", the teachings of which are incorporated herein.

The peptides or proteins of this invention can be administered asmultivalent subunit vaccines in combination with other antigens of H.influenzae. For example, they may be administered in conjunction witholigo- or polysaccharide capsular components of H. influenzae such aspolyribosylribitolphosphate (PRP).

As mentioned, peptides and proteins having epitopes of protein "e" evokebactericidal antibodies which act synergistically in killing H.influenzae with antibodies against other outer membrane proteins of H.influenzae. Thus, in an embodiment of the invention, protein "e" (or apeptide or protein having a common epitope) is administered inconjunction with other outer membrane proteins of H. influenzae (orpeptides or proteins having epitopes thereof) to achieve a synergisticbactericidal activity. Particularly preferred outer membrane proteins ofH. influenzae are the 15,000-dalton peptidoglycan-associated outermembrane lipoprotein (PAL) and the 15,000-dalton Haemophilus lipoproteinPCP described by Deich, R. A. et al. (1988) J. Bacteriol.170(2):489-498, the teachings of which are incorporated by referenceherein. For combined administration with epitopes of other outermembrane proteins, the protein "e" peptide can be administeredseparately, as a mixture or as a conjugate or genetic fusion peptide orprotein. For example, the PAL and PCP or any proteins, peptides orepitopes derived from them, can be administered as a mixture or as aconjugate or fusion with a protein "e" or a protein "e" derived peptideor protein. The conjugates can be formed by standard techniques forcoupling proteinaceous materials. Fusions can be expressed from fusedgene constructs prepared by recombinant DNA techniques as described.

Protein "e" or any derived peptides or proteins can be used inconjunction with antigens of other organisms (e.g. encapsulated ornonencapsulated, bacteria, viruses, fungi and parasites). For example,protein "e" can be used in conjunction with antigens of othermicroorganisms implicated in otitis media. These include Streptococcuspneumoniae, Streptococcus pyogenes, group A, Staphylococcus aureus,respiratory syncytial virus and Branhamella catarrhalis.

In formulating the vaccine compositions with the peptide or protein,alone or in the various combinations described, the immunogen isadjusted to an appropriate concentration and formulated with anysuitable vaccine adjuvant. Suitable adjuvants include, but are notlimited to: surface active substances, e.g., hexadecylamine,octadecylamine, octadecyl amino acid esters, lysolecithin,dimethyl-dioctadecylammonium bromide), methoxyhexadecylgylcerol, andpluronic polyols; polyamines, e.g., pyran, dextransulfate, poly IC,carbopol; peptides, e.g., muramyl dipeptide, dimethylglycine, tuftsin;oil emulsions; and mineral gels, e.g., aluminum hydroxide, aluminumphosphate, etc. and immune stimulating complexes. The immunogen may alsobe incorporated into liposomes, or conjugated to polysaccharides and/orother polymers. For use in a vaccine formulation.

The vaccines can be administered to a human or animal in a variety ofways. These include intradermal, intramuscular, intraperitoneal,intravenous, subcutaneous, oral and intranasal routes of administration.

Live vaccines

The peptide and proteins of this invention can be administered as livevaccines. To this end, recombinant microorganisms are prepared thatexpress the peptides or proteins. The vaccine recipient is inoculatedwith the recombinant microorganism which multiplies in the recipient,expresses the protein "e" peptide or protein and evokes a immuneresponse to H. influenzae. Preferred live vaccine vectors are poxviruses such as vaccinia (Paoletti and Panicali, U.S. Pat. No.4,603,112) and attenuated Salmonella strains (Stocker, U.S. Pat. No.4,550,081).

Live vaccines are particularly advantageous because they lead to aprolonged stimulus which can confer substantially long-lasting immunity.When the immune response is protective against subsequent H. influenzaeinfection, the live vaccine itself may be used in a preventative vaccineagainst H. influenzae.

Multivalent live vaccines can be prepared from a single or a fewrecombinant microorganisms that express different epitopes of H.influenzae (e.g., other outer membrane proteins such as PAL and PCP orepitopes thereof). In addition, epitopes of other pathogenicmicroorganisms can be incorporated into the vaccine. For example, avaccinia virus can be engineered to contain coding sequences for otherepitopes in addition to those of H. influenzae. Such a recombinant virusitself can be used as the immunogen in a mulivalent vaccine.Alternatively, a mixture of vaccinia or other viruses, each expressing adifferent gene encoding for different epitopes of outer membraneproteins of H. influenza and/or epitopes of other disease causingorganisms can be formulated as a multivalent vaccine.

An inactivated virus vaccine may be prepared. Inactivated vaccines are"dead" in the sense that their infectivity has been destroyed, usuallyby chemical treatment (e.g., formaldehyde treatment). Ideally, theinfectivity of the virus is destroyed without affecting the proteinswhich carry the immunogenicity of the virus. In order to prepareinactivated vaccines, large quanitites of the recombinant virusexpressing the desired epitopes are grown in culture to provide thenecessary quantity of relevant antigens. A mixture of inactivatedviruses express different epitopes may be used for the formulation of"multivalent" vaccines. In certain instances, these "multivalent"inactivated vaccines may be preferable to live vaccine formulationbecause of potential difficulties arising from mutual interference oflive viruses administered together. In either case, the inactivatedvirus or mixture of viruses should be formulated in a suitable adjuvantin order to enhance the immunological response to the antigens. Suitableadjuvants include: surface active substances, e.g., hexadecylamine,octadecyl amino acid esters, octadecylamine, lysolecithin,dimethyl-dioctadecylammonium bromide, N, N-dicoctadecyl-N'-N'bis(2-hydroxyethyl-propane diamine), methoxyhexadecylglycerol, and pluronicpolyols; polyamines, e.g., pyran, dextransulfate, poly IC, carbopol;peptides, e.g., muramyl dipeptide, dimethylglycine, tuftsin; oilemulsions; and mineral gels, e.g., aluminum hydroxide, aluminumphopshate, etc.

Passive Immunity and Anti-Idiotypic Antibodies

The bactericidal antibodies induced by protein "e" epitopes can be usedto passively immunize an individual against H. influenzae. Passiveimmunization confers short-term protection for a recipient by theadministration of the pre-formed antibody. Passive immunization can beused on an emergency basis for special risks, e.g., young childrenexposed to contact with bacterial meningitis patients.

The peptides and proteins of this invention can be used to producepolyclonal or monoclonal antibodies for use in passive immunotherapyagainst H. influenzae. Human immunoglobulin is preferred becauseheterologous immunoglobulin may provoke a deleterious immune response toits foreign immunogenic components. Polyclonal antisera can be obtainedfrom individuals immunized with the peptides or proteins in any of theforms described. Immunoglobulin fraction can then be enriched. Forexample, immunoglobulins specific for epitopes of protein "e" can beenriched by immunoaffinity techniques employing the peptides or proteinsof this invention. The antibody can be specifically adsorbed fromantisera onto an immunoadsorbent containing epitopes of protein "e" andthen eluted from the immunoadsorbent as an enriched fraction ofimmunoglobulin.

Monoclonal antibodies against epitopes of protein "e" can be made bystandard somatic cell fusion techniques of Kohler and Milstein, Nature256:495 (1975) or similar procedures employing different fusing agents.Briefly, the procedure is as follows: an animal is immunized withprotein "e" or immunogenic fragments or conjugates thereof. For example,haptenic oligopeptides of protein "e" can be conjugated to a carrierprotein to be used as an immunogen. Lymphoid cells (e.g. spleniclymphocytes) are then obtained from the immunized animal and fused withimmortalizing cells (e.g. myeloma or heteromyeloma) to produce hybridcells. The hybrid cells are screened to identify those which produce thedesired antibody.

Human hybridomas which secrete human antibody can be produced by theKohler and Milstein technique. Although human antibodies are especiallypreferred for treatment of human, in general, the generation of stablehuman-human hybridomas for long-term production of human monoclonalantibody can be difficult. Hybridoma production in rodents, especiallymouse, is a very well established procedure and thus, stable murinehybridomas provide an unlimited source of antibody of selectcharacteristics. As an alternative to human antibodies, the mouseantibodies can be converted to chimeric murine/human antibodies bygenetic engineering techniques. See V. T. Oi et al., Bio Techniques 4(4):214-221 (1986); L. K. Sun et al., Hybridoma 5 (1986).

The monoclonal antibodies specific for protein "e" epitopes can be usedto produce anti-idiotypic (paratopespecific) antibodies. See e.g.,McNamara et al., Dec., 14, 1984, Science, page 1325; Kennedy, R. C. etal., (1986) Science 232:220. These antibodies resemble the protein "e"epitope and thus can be used as an antigen to stimulate an immuneresponse against H. influenzae.

Digaynostic Assays

The peptides and proteins of this invention may be used as antigens inimmunoassays for the detection of H. influenzae in various tissues andbody fluids e.g., blood, spinal fluid, sputum, etc. A variety ofimmunoassay systems may be used. These include: radioimmunoassays, ELISAassays, "sandwich" assays, precipitin reactions, gel diffusionprecipitin reactions, immunodiffusion assays, agglutination assays,fluorescent immunoassays, protein A immunoassays andimmunoelectrophoresis assays.

In addition, nucleic acids having the nucleotide sequence of the geneencoding protein "e" (FIG. 6) or any nucleotide sequences whichhybridize therewith can be used as probes in nucleic acid hybridizationassays for the detection of H. influenzae in various tissues or bodyfluids of patients. The probes may be used in any nucleic any type ofhybridization assay including: Southern blots (Southern, 1975, J. Mol.Biol. 98:508); Northern blots (Thomas et al., 1980, Proc. Nat'l Acad.Sci. USA 77:5201-05); colony blots (Grunstein et al., 1975, Proc. Nat'lAcad. Sci. USA 72:3961-65), etc. Stringency of hybridization can bevaried depending on the requirements of the assay.

The invention is further illustrated by the following examples.

Exemplification

I. Isolation of protein "e"

Isolation of Haemophilus Cell Envelopes

Cell envelopes were isolated from Hib strain Eagan cells grown on eitherbrain heart infusion medium containing 10 μg/ml hemin and 1 μg/ml NAD(BHI/XV) or mMIC (modification of Herriott et al., J. Bacteriol.,101:513-516 (1970)) media. Cells were harvested from liquid cultures bycentrifugation at 10,000×g, 4° C. For 10 minutes. The cell pellet wasweighed and resuspended in 10 mM HEPES-NaOH (pH 7.4), 1 mM EDTA, with avolume equal to five times the wet weight of the cells. The cells weredisrupted using a Gaulin homogenizer. The disrupted cell suspension wascentrifuged at 10,000×g for 5 minutes at 4° C. to remove unbroken cellsand large debris. The supernatant fraction was saved and NaCl added to0.5 M. Cell membranes were pelleted by centrifugation at 100,000×g forabout 1 h at 4° C.

An outer membrane-cell wall complex was obtained by removing the innermembranes from the total membrane fraction by repeated extraction of thetotal membrane fraction with 2% Triton X-100 in 10 mM HEPES-NaOH, 1 mMMgCl₂, pH 7.4. The insoluble residue containing the outer membrane-cellwall complex was pelleted by centrifugation at 350,000×g for 30 minutesat 4° C. This complex was then resuspended in 50 mM Tris-HCl, 5 mM Na₂EDTA, pH 8 and stored at 4° C.

Isolation of Protein "e" from Haemophilus Cell Envelopes

Contaminating proteins were solubilized from H. influenzae cellenvelopes by differential detergent extraction as follows. Cellenvelopes prepared as described above were sequentially extracted twicewith 1% sarcosyl in 50 mM Tris-HCl, 5 mM Na₂ EDTA, pH 8 and theinsoluble material recovered by centrifugation at 350,000×g for 30minutes at 20° C., then twice with 1% Zwittergent™ 3-12 in the samebuffer, 50 mM Tris-HCl, 5 mM Na₂ EDTA, pH 8. The protein "e" was nowsolubilized from the insoluble residual outer membrane-cell wallmaterial by extraction with 1% Zwittergent™ 3-14 in 50 mM Tris-HCl, 5 mMNa₂ EDTA, pH 8. This extraction was repeated three times. Thesolubilized protein "e" containing fractions were pooled and passedthrough a DEAE column equilibrated with 50 mM Tris-HCl, 5 mM Na₂ EDTA,pH 8. The protein "e" was not retained under these conditions but themajor protein contaminants were retained. The fall-through fractionscontaining protein "e" were then passed over a hydroxylapatite columnwhich had been equilibrated with 50 mM Tris-HCl, pH 8. The protein "e"was retained under these conditions. The hydroxylapatite column with theadsorbed protein "e" was then washed with one column volume of 50 mMTris-HCl, pH 8. The protein "e" was eluted from the hydroxylapatite with1% Zwittergent™ 3-14 in 0.3 M dibasic phosphate, pH 8. Fractionscontaining protein "e" were pooled, concentrated by diafiltration, andprecipitated with ethanol. The precipitated protein "e" was thensolubilized again by differential detergent extraction. The precipitatewas first extracted with 1% octylglucoside in 50 mM Tris-HCl, pH 8 andthe insoluble protein "e" remained in the precipitate. The protein "e"was then solubilized with 1% Zwittergent™ 3-14 in 50 mM Tris-HCl, 5 mMNa₂ EDTA, pH 8. More preferably, the fall through fractions from theDEAE column were passed over an S Sepharose™ (Pharmacia) fast flowcolumn (cation exchange column), previously equilabrated with 50 mMTris-HCl, 5 mM Na₂ EDTA (pH8), containing 0.1% Zwittergent™ 3-14. Theprotein "e" adsorbed to the column and was eluted with a 0-0.5 M NaClgradient in the same buffer. Protein "e" prepared as described above issubstantially pure and essentially free of endotoxin could be furtherconcentrated as previously described or used as eluted.

Characterization of protein "e" by Amio Acid Composition and Sequence

Amino acid analysis was performed according to the procedure of Simpsonet al. (J. Biol. Chem., 251:1936-1940 (1976)). Hydrolysis wasaccomplished by heating 0.5-1 mg of protein in 0.1 ml of 4 N methanesulfonic acid under vacuum in a thick-walled sealed glass tube at 150°C. for 90 minutes. The quantity of each amino acid is obtained bycomparison of the areas under the various peaks with areas obtainedusing known quantities of standard amino acids. Results obtained areillustrated in Table 1.

Samples were prepared for analysis by SDS-PAGE by adjusting them to 0.1M Tris-HCl, pH 7.0, 25 mM dithiothreitol, and 2% SDS with 5×concentrated sample buffer, then heating for 5 minutes at 100° C. Priorto electrophoresis all samples were adjusted to 6% (w/v) sucrose and0.001% bromophenol blue. Most routine analyses were performed using theBio-Rad Mini Protein Gel System (Redmond, Calf.). Gels were 1.5 mm thickand the separating gel contained 15% acrylamide with an acrylamide tobis ratio of 30:0.8, 0.375 M Tris-HCl (pH 8.8) and 0.1% SDS. Thestacking gel contained 4.8% acrylamide with the same ratio of acrylamideto bis, 125 mM Tris, HCl (pH 7.0), and 0.1% SDS per gel. Followingelectrophoresis gels were stained for at least 1 hour with 0.125% (w/v)Coomasie blue in ethanol: acetic acid: water (5:1:5), then destained inthe same solvent system without the blue dye. Pre-stained low molecularweight standards which included the following: ovalbumin, 43,000;alpha-chymotrypsinogen, 25,700; Beta-lactoglobulin, 18,400; lysozyme,14,300; bovine trypsin inhibitor, 6,200; insulin (A and B Chains), 2,300and 3,400 (BRL, Bethesda, Md.) were used to assist in the determinationof the relative molecular weight of the protein "e".

Further purification of protein "e" can be achieved by standard methodssuch as ion exchange chromatography, molecular sieving, hydrophobic orreserve phase chromatography, chromatofocusing, gel electrophoresis andthe like.

Substantially pure protein "e" was analyzed in an SDS-PAGE system todetermine the relative molecular weight of the reduced and denaturedform of the protein and to assess its purity (FIG. 1). A sample purified"e" protein (3 μg) was analyzed in a 15% SDS-FAGE system and stainedwith Coomassie blue. Lane 1; purified "e" protein; Research LaboratoriesLife Technologies, Inc., which included ovalbumin, α-chymotrypsinogen,β-lactoglobulin, lysozyme, bovine trypsin inhibitor, and insulin (A andB chains). The reported respective molecular weights of the standardsand 43,000; 25,700; 18,400; 6,200; 2,300 and 3,400.

                  TABLE 1    ______________________________________    AMINO ACID COMPOSITION OF    THE H. INFLUENZAE "e" PROTEIN                 Analysis    Amino Acid   Methane Sulfonic Acid    ______________________________________    Asp                   34       (38)    Thr                   9        (8)    Ser                   9        (10)    Glu                   29       (29)    Pro                   6        (4)    Gly                   25       (24)    Ala                   29       (28)    Cys*                  0        (1)    Val                   18       (18)    Met*                  5        (6)    Ile                   7        (5)    Leu                   17       (14)    Tyr                   11       (12)    Phe                   12       (12)    His                   4        (4)    Lys                   26       (26)    Trp                   nd       (6)    Arg                   11       (9)    ______________________________________     Values have been adjusted to nearest whole number and are expressed in     terms of residues/proteins.     *Values given for these amino acids are from the respective digests. None     of the forms for cysteine were observed even after performing acid     oxidation, however, five (5) methionyl residues would be predicted using     performic acid as well as methanesulfonic acid. Residues in () are     predicted from the available DNA sequence and peptide mapping.

Initial attempts at sequencing protein "e" using Edman chemistry failedto yield satisfactory results because of a blocked N-terminal residue.In order to obtain partial amino acid sequence information, it wasnecessary to enzymatically digest protein "e" with proteolytic enzymesto obtain peptide fragments that were amenable to sequence analysis.

Samples of the "e" protein (0.3 mg/mL) were incubated overnight with oneof three proteases, endoproteinase Lys-C, Arg-C, or V8, at an enzyme toprotein ratio of 1:100 at 37° C. Peptides from these endoproteinasedigests were obtained by reverse phase HPLC analysis. Samples (50-100μL) of each of the digests were analyzed on an Aquapore RP-300 column onthe Applied Systems microbore HPLC with the detection wavelength set at220 nm. Buffer A was 0.1% TFA and Buffer B was 95% acetonitrile in 0.1%gradient up to 40% Buffer B at 15 min, then a steeper gradient to 100%Buffer B at 17.5 min and continuing at 100% B for 2.5 min more.Fractions were collected by hand. Amino acid sequencing was performedaccording to the manufacturer's instructions on the Applied Biosystemsprotein sequinator. The results are shown in Table 2; ? indicate cycleswhere no residue could be assigned.

                  TABLE 2    ______________________________________    AMINO ACID SEQUENCES OF PEPTIDES DERIVED FROM    ENDOPROTEINASE DIGESTION OF THE "e" PROTEIN    ______________________________________    ELys#1  A-R-L-D-A-V-Q-A-W-D-G-K    ELys#2  R-L-G-F-N-G-V-E-E-S-A-F-Y-L-K    ELys#4a T-F-I-M-L-P-N-A-N-Y-G-G-W-E-G-G-L-A-E-G-Y-            F-K    ELys#4b A-V-V-A-D-L-D-E-T-M-L-D-N-?-P-Y-?-?-W-Q-V-            ?-N-?-?-?-F-D-G-K    ELys#5  S-E-E-H-A-N-M-Q-L-Q-Q-Q-A-V-L-G-L-N-W-M-Q-            D-S-G    EArg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

Western blot analysis of globomycin-treated recombinant E. coliexpressing protein "e" revealed the presence of two "e" reactive bands.Globomycin inhibits the action of signal peptidase II which cleavessignal peptides having bacterial lipidation signals. Thus, the protein"e" behaves as expected of a lipoprotein.

II. Preparation of Anti-protein "e" Antibodies

Preparation of Polyclonal Anti-protein "e" Antiserum

Substantially pure protein "e" was used as an immunogen to prepareanti-protein "e" antibodies. The protein "e" was isolated as describedabove and was mixed with incomplete Freund's adjuvant and emulsified.Rabbits were injected intramuscularly with approximately 50 μg ofprotein "e" in the adjuvant mixture. Animals were reimmunizedapproximately 4 weeks and 8 weeks after the initial immunization andbled one week following the final immunization.

Production of Anti-protein "e" Monoclonal Antibodies

Hybridoma cell lines secreting antibodies to protein "e" were obtainedby fusion of mouse myeloma cell line P3XAg.653 with spleen cells from aBalb/c mouse immunized against protein "e" as follows. Mice wereimmunized intraperitoneally at 8 weeks of age with approximately 10 μgof protein "e" enriched OMPs (see above) emulsified in incompleteFreund's adjuvant. Two weeks later, mice were boosted with the identicalvaccine preparation. Mice were boosted again on week 5 with 1 μg of theprotein "e" enriched outer membrane proteins (OMPs) in saline injectedintravenously. Three days after the last immunization, the mice weresacrificed and spleen cells isolated for fusion. Culture supernatantsfrom fused cells were screened for activity against the protein "e"enriched OMP preparation by ELISA. Positive cultures were then screenedfor activity against total OMPs of Haemophilus by Western blot. Culturesreactive with a band of approximately 28,000 daltons were clonedby.limiting dilution. The resulting clones were rescreened for positivesecretion against purified protein "e". Cell lines were tested foractivity against E. coli OMPs and against lipooligosaccharide (LOS) ofH. influenzae by Western blot. Cell lines which were positive for theprotein "e" and negative against both E. coli OMPs and Haemophilus LOSwere saved.

Binding specificities of monoclonal antibodies from three selectedsecreting cell lines were determined by competitive radioimmunoassay.Monoclonal antibodies were intrinsically labeled with ³ H by addition of³ H-leucine to growth media. Labeled antibodies were used in solid phaseradioimmunoassay in competition with unlabeled antibodies for binding toprotein "e". Monoclonal antibody EPR 5.2.1 did not compete with theother two antibodies and recognizes a distinct epitope. Monoclonals EPR17.1 and EPR 35.24 show some competition with each other, but do notcompletely block each others binding. Thus, these two antibodiesrecognize epitopes, which either overlap or have some steric hindrancewhen bound to protein "e".

III. Reactivity of Anti-protein "e" Antibodies Against ClinicalNontypable H. Influenza Isolates

Monoclonal and polyclonal anti-protein "e" antibodies were testedagainst whole cell isolates of clinical nontypable strains. Clinicalstrains were grown overnight in BHI-XV and aliquots of each culture wereseen on SDS-PAGE gels and their reactivity with anti-protein "e"antibodies examined by immunoblot analysis. The results of immunoblotanalysis with polyclonal anti-protein "e" antiserum indicated thatprotein "e" is recognized by the anti-protein "e" antiserum in everystrain.

The results of immunoblot analysis of clinical isolates with monoclonalantibodies, Mab EPR 5.2.1 and Mab EPR-17.2.1, are shown in FIGS. 2a and2b, respectively. Each monoclonal antibody recognizes a differentepitope on protein "e". All of the monoclonals react with protein "e" ineach clinical isolate. Thus, epitopes on protein "e" are conserved amonga variety of clinical nontypable isolates.

IV. General Procedures Used for Preparation of Recombinant Plasmids

Conditions for Restriction Enzyme Digestions

Restriction endonucleases were purchased from BRL (Bethesda, Md.) IBI(New Haven, Conn.), New England Biolabs (Beverly, Mass.) or USBiochemical (Cleveland, Ohio).

Restriction enzyme digestions were carried out by suspending DNA in theappropriate restriction buffer, adding restriction endonuclease andincubating for an appropriate period of time to ensure completedigestion. One unit of enzyme is defined as the amount required tocompletely digest 1.0 μg of phage lambda DNA in 1 hour in a totalreaction mixture of 20 μl volume. Buffers used with the various enzymesare listed below:

Low salt buffer used for ClaI, HpaI, and KpnI digestions consisted of:10 mM Tris (pH 8.0), 10 mM MgCl₂ and 10 mM dithiothreitol (DTT).

Medium salt buffer used for AvaI, EcoRV, HaeII, HincII, HindIII, PstI,SphI, SspI, and XhoI digestions consisted of: 50 mM Tris (pH 8.0), 10 mMMgCl₂, 50 mM NaCl, and 10 mM DTT.

High salt buffer used for BamHI, EcoRI, PvuI, SalI and XbaI digestionsconsisted of: 50 mM Tris (pH 8.0), 10 mM MgCl₂, 150 mM NaCl and 10 mMDTT.

The buffer used for SmaI digestions consisted of: 10 mM Tris (pH 8.0),20 mM KCl, 10 mM MgCl₂, and mM DTT. All restriction digestions werecarried out at 37° C. except TaqI which was carried out at 60° C. andSmaI which was carried out at 25° C.

Gel Purification of DNA Fragments

After restriction enzyme digestions, DNA fragments of varying sizes wereseparated and purified using gel electrophoreses in low meltingtemperature agarose (FMC LGT agarose) using 50 mM Tris-acetate 1 mM EDTAbuffer pH 7.8 at 10 volts/cm. Agarose concentrations varied from 0.8% to1.5% depending on the size of fragments to be recovered. DNA bands werevisualized by ethidium bromide fluorescence and cut out of the gel. DNAwas recovered by melting the agarose at 65° C., adding 4 volumes of 0.65M NaCl, 10 M Tris (pH 8.0), 1 mM EDTA to bring the mixture to a finalconcentration of 0.5. M NaCl, loading the DNA onto a NACS column (BRL,Bethesda, Md.) equilibrated with 0.5 mM NaCl, 10 mM Tris pH 8.0, 1 mMEDTA (loading buffer), washing the column with 3-5 volumes of loadingbuffer, and eluting with 2-3 volumes 2 m NaCl, 10 mM Tris pH 8.0, 1 mMEDTA. The DNA eluate was diluted 1:1 with double distilled H2O andprecipitated with 3 volumes of ethanol. The pellet was washed with 70%ethanol, vacuum dried, and resuspended in 10 mM Tris-HCL buffer, pH 7.5containing 1 mM EDTA (TE buffer).

DNA Ligation

All legations were accomplished using T4 DNA ligase. T4 DNA ligase waspurchased from BRL (Bethesda, Md.), United States Biochemicals(Cleveland, Ohio) or Boehringer (Indianapolis, Ind.). One unit (U) of T4DNA ligase is defined as the amount required to yield 50% ligation ofHindIII fragments of bacteriophage lambda DNA in 30 minutes at 16° C. in20 μl volume ligase buffer at a 5'-DNA termini concentration of 0.12 μM(300 μg/ml). DNA ligations were performed in ligase buffer consistingof: 50 mM Tris (pH 7.5), 10 mM MgCl2, 10 mM DTT, 1 mM adenosinetriphosphate). Normally a DNA concentration ranging from 2-30 μg/ml, anda molar ratio of vector to insert of 1:2 was used. T4 DNA ligase wasadded at a ratio of 1 U per 20 μl reaction volume.

Incubations were carried out for 18-24 hours. Temperatures used were 15°C. for cohesive end ligations, and 22° C. for blunt end legations. Ifsufficient material was available, ligations were checked by analyzing aportion of the reaction mixture by agarose gel electrophoresis.

Protein Immunoblot Analysis (Western Blot)

Proteins were fixed to nitrocellulose sheets for immunoblot analysis byvarious techniques, depending on the particular application. Phageplaques were transferred from agar plates by gently placing a sterile8.1 cm diameter nitrocellulose disc onto the surface of a 10 cm diameterphage titer plate. The sheet was allowed to wet completely, positionswere marked by punching through the filter with a sterile needle, andthe filter was lifted after two minutes.

Colony blots were performed by transferring bacterial colonies to anitrocellulose sheet, allowing the colonies to grow by placing the sheet(colony side up) on nutrient agar for 4 to 6 hours, and exposing. Thesheet to chloroform vapor for 30 minutes to lyse the colonies.

Protein gel transfers were performed by placing an SDS-PAGE gelcontaining the protein mixture to be analyzed on a nitrocellulose sheetand applying horizontal electrophoresis in a Hoeffer Transphor apparatusat 0.5 A for 14 hours in 25 mM Tris. 0.38 M glycine pH 8.8 buffer.

Once protein transfer was complete, filters were soaked in 50 mM Tris(pH 8.0), 150 mM NaCl, 5% nonfat dry milk (BLOTTO) at 37° C. for onehour in all cases, except colony blots. When colony blots wereperformed, the filters were soaked overnight at 4° C. in BLOTTOcontaining 1 mg/ml lysozyme to digest cell debris. Filters were thenabsorbed with a first antibody probe at an appropriate dilution(determined by trial and error) in BLOTTO for 3 hours at 37° C., washedthree times for 15 minutes with BLOTTO, absorbed with horseradishperoxidase conjugated second antibody (Kirkegaard and Perry,Gaithersburg, Md.) at a dilution of 1:500 in BLOTTO for one hour at 37°C. and washed with BLOTTO three times for 15 minutes. Filters wereplaced in 50 mM Tris (pH 7.0), 150 mM NaCl, 0.01% hydrogen peroxide; and0.06% 4-Chloro-1-naphthol (Sigma Chemical Co., St. Louis, Mo.) inmethanol was added. If no blue color developed within 20 minutes, thereaction was considered negative. The reaction was stopped bytransferring the filter to distilled water and blotting dry.

Dry Filter Hybridization Analysis (Southern Blot)

DNA filter hybridization analysis was carried out according to themethod of Smith and Summers (Anal. Biochem., 109:123-129 (1980)). DNA tobe analyzed was digested with the appropriate restrictionendonuclease(s) and separated by agarose gel electrophoresis in 0.7%agarose (SeaKem, FMC, Rockland, Me.) using 89 mM Tris, 89 mM borate, 8mM EDTA buffer at 1.5 V/cm. DNA in the gel was depurinated by treatmentwith 0.25 m HCl for 15 minutes and then denatured in 0.5 M NaOH, 1.5 MNaCl for a total of 30 minutes. The gel was neutralized with 1 Mammonium acetate, 0.02 M NaOH for 1 hour and the DNA transferredbidirectionally to nitrocellulose (BA85, Schleicher and Scheull, Keene,N.H.) in the above buffer by paper blotting. After the transfer wascomplete, approximately 1 hour, the filters were removed, lanes markedwith ink, and rinsed in 2× SSC (prepared by dilution from a 20× stocksolution containing 175.6 g NaCl and 88.2 g Na citrate per liter) andair dried. DNA fragments were fixed to filters by baking at 80° C. For 2hours under vacuum.

Probes for DNA hybridization were prepared using a nonradioactive DNAlabeling and detection kit purchased from Boehringer-Mannheim(Indianapolis, Ind.). Probe DNA was linearized with an appropriaterestriction endonuclease, extracted with a 1:1 mixture ofphenol:chloroform, and precipitated with ethanol. The DNA precipitatewas dissolved in 10 μl of 10 mM Tris, 1 mM EDTA, pH 8.0 (TE) anddenatured by heating to 95° C. for 10 minutes. DNA was quickly cooled indry ice/ethanol and transferred to ice. The DNA was labeled using therandom hexanucleotide mix supplied with the kit as primer, the labelingmixture provided which includes digoxigenin-dUTP (dig-dUTP), and theKlenow fragment of E. coli polymerase I. After the reaction mixture wasincubated for 18 hours at 37° C., the reaction was stopped by additionof 1 μ1 of 0.5 M EDTA, pH 8.0. Twenty μg of yeast tRNA was added ascarrier and the DNA precipitated with ethanol. Three μg of template DNAyielded approximately 0.5 μg of labeled DNA.

Filters to be probed were rehydrated in deionized water, and incubatedat 68° C. in a solution containing 5× SSC, 0.5% blocking reagent(supplied in the kit), 0.1% N-laurylsarcosine, 0.02% SDS for 6 hours.The hybridization solution consisted of the above buffer with 30 ng oflabeled probe DNA per ml at a ratio of 2.5 ml per 100 cm² of filter..The probe solution was denatured by heating to 95° C. for 10 minutes andadded to the filters. Hybridizations were done at 68° C. for 18 hours.Filters were washed 1× in 2× SSC, 0.1% SDS at room temperatures, and2×15 minutes in 0.1× SSC, 0.1 SDS at 68° C. After air drying, hybridizeddig-dUTP containing probe was detected using the supplied alkalinephosphatase conjugated anti-digoxigenin antisera at a 1:5000 dilutionand development of the alkaline phosphatase-nitrobluetetrazolium-5-bromo-4-chloro-3-indoyl phosphate color reaction for 2-4hours. The reaction was stopped by rinsing the filter in TE. Under theabove conditions, DNA homologies of greater than 98% would show positivebinding of the labeled probe.

V. Isolation of protein "e" Gene

The amplified phage library prepared as described in section 6.5.1 wasdiluted to 10⁻³ PFU per ml in TMC and 100 μl of E. coli KH802 (5×10cells/ml) were added. After incubation at 37° C. for 20 minutes, 3 ml ofLB media containing 10 mM MgCl₂ and 0.85% agar at 56° C. were added andthe suspension plated onto LB agar plates containing 10 mM MgCl₂. Plateswere incubated overnight at 37° C. to allow plaque formation, andchilled to 4° C. Plaques were transferred to nitrocellulose filters byabsorption and the filters were probed with a pool of monoclonalantibodies which react with protein "e" as described above. Severalpositive plaques were identified in this manner. Positive plaques werepicked and the phage allowed to elute into 1 ml of TMG at 4° C. Thephage were amplified by growth in E. coli KH802. Clones were verified byscreening phage lysates with SDS-PAGE/Western blot techniques usinganti-protein "e" monoclonal antibodies as probes. Positive clonesexpressed a protein of apparent molecular weight 30,000 daltons whichreacted with the anti-protein "e" monoclonal antibodies. This proteinwas not present in control lysates of negative plaques.

One positive plaque designated EP1-1 was chosen for further analysis.This phage isolate was amplified by growth in E. coli KH802 in LB brothcontaining 10 mM MgCl₂ and the phage particles reocvered byprecipitation with 20% polyethylene glycol 6000 and banding in CsCl stepgradient (See Maniatis et al., supra). Phage DNA was isolated bytreatment with 0.1% SDS proteinase K (10 μg/ml, Sigma Chemical Co., St.Louis, Mo.) at 65° C. for 2 h followed by extraction with an equalvolume of phenol, then an equal volume of chloroform. The DNA wasprecipitated by addition of ammonium acetate to 2 M and 2.5 volumes ofice-cold ethanol. After incubation at -20° C., the DNA was pelleted bycentrifugation at 13,000×g.

Phage DNA was digested with EcoRI to separate insert fragments from theλ arms. When digested DNA was electrophoresed on a 0.6% agarose gel, asingle band of approximately 15 Kb was observed in addition to the λarms. This 15 Kb fragment was subcloned into the EcoRI site of pUC18.The resulting clones expressed a protein reactive with the anti-protein"e" monoclonal antibodies and of identical molecular size with thenative protein "e" as determined by SDS-PAGE/Western blot analysis (FIG.3). The 15 Kb insert fragment in this plasmid, pPX504, was digested withSspI to delete excess DNA and ligated with SspI-HincII fragment ofpUC18. The resulting plasmid, pPX513 (FIG. 4) contained anEcoRI-SspI/HincII insert fragment of approximately 1.6 Kb and expresseda protein which reacted with monoclonal antibodies against protein "e"under regulation of the native Haemophilus promoter (FIG. 5).

VII. Determination of the Nucleotide Sequence of protein "e" Gene

The nucleotide sequence of the protein "e" gene of pPX513 was determineddirectly from the plasmid by dideoxynucleotide sequencing using thedouble stranded plasmid as template (Zagursky et al., Tabor et al.,supra). M13, M18 and M19 clones of the EcoRI-SspI fragment of pPX513were sequenced. All sequencing. primers were made at Praxis Biologics,Rochester, N.Y. on an Applied Biosystems 380B DNA synthesizer. Theprimers were made on a 0.2 μmole controlled pore glass column withbeta-cyanoethyl phosphate protecting group chemistry. The yield ofoligonucleotide was sufficiently pure to allow the use of the primersdirectly from the column without further purification. The entiresequence of the gene is shown in FIG. 6. This ORF encodes a polypeptideof 274 amino acids. The deduced amino acid sequence of protein "e" isshown in FIG. 7. The amino acid composition of the deduced protein "e"closely matches that of purified protein "e" (Table 2). The protein "e"gene also has an internal peptide-sequence (AA) which aligns with thesequence of peptide L5. The amino terminal residue peptide resembles amembrane transport signal sequence determined for other proteins(Watson, 1984, supra). Thus we conclude that this gene encodes theprotein "e".

Chromosomal DNA was prepared from E. coli and H. influenzae strainsHDG-85 and Eagan by the method of Marmur (J. Mol. Biol., 3:208-218(1962)). The DNA was cut with EcoRI and Southern blots prepared asabove. These blots were probed with a dig-dUTP labeled protein "e" geneclone prepared as described above. Results are shown in FIG. 8. Theprobe recognized a single band of approximately 15 Kb in each H.influenzae chromosome and did hybridize to either the lambda standardsor the E. coli chromosome showing that the cloned gene is a Haemophilusgene and that it is carried in a single copy.

VIII. Bactericidal Activity of protein "e" Subunit Vaccine

Anti-protein "e" polyclonal antisera, prepared as described, wereexamined for their ability to kill Hi in an in vitro complement mediatedbactericidal assay system (see Musher et al, Infect. Immun., 39:297-304(1983); Anderson et al., J. Clin. Invest., 51:31-38 (1972)).Bactericidal assays were performed using precolostral calf serum (PCCS),stored at -70° C., as complement source. The PCCS was prepared for usein the bactericidal (BC) assay by adsorption with whole cells of thenontypable H. influenzae strain being tested. A one milliliter aliquotof an overnight culture grown in BHI-XV was pelleted by centrifugationin an Eppendorf table top centrifuge. The pellets were washed byresuspending in sterile phosphate buffered saline containing 0.15 mMCaCl₂ and 0.5 mM MgCl₂ (PCM) and repeating the centrifugation. Onemilliliter of PCCS was thawed and used to resuspend the bacterialpellet. The sample was held on ice for one hour. The bacteria wereremoved by centrifugation, and a second bacterial pellet was resuspendedin the PCCS. This was held on ice for one hour. The sample wascentrifuged to remove the bacteria and then filter sterilized with a0.22 micron membrane. The prepared PCCS was held on ice until used.Bacteria were prepared by diluting overnight cultures 1:15 in BHI-XVbroth and by incubating at 37° C. with aeration. Cells were grown to anoptical density of 0.9 at 490 nm (approximately 10⁹ CFU/ml). Bacteriawere diluted 40,000 fold in sterile PCX with 0.5% bovine serum albumin(PCMA). The final dilution contained 25% PCCS (v/v). Immunoglobulinsfrom polyclonal mouse anti-protein "e" antiserum were precipitated withsaturated ammonium ammonium sulfate at 35% final concentration at 4° C.overnight. The precipitate was collected by centrifugation for 10minutes at 4° C. in an Eppendorf centrifuge. The supernatant wasdiscarded and the pellet resuspended in PCM at 10 times original volume.The sample was restored to original volume using a Centriconmicroconcentrator unit with a 10,000 molecular weight cut off membrane.The sample was washed in PCM an additional four times as described aboveto remove residual ammonium sulfate. Polyclonal rabbit sera were notpretreated for use in the BC assay.

Fifteen microliters of the serum sample were placed in the first well ofa sterile 96 well U-bottom microtiter plate held on ice. Two-fold serialdilutions using PCMA were done in the remaining wells. The plates wereremoved from the ice and 15 μl of the cell/complement mixture were addedto the serum in the wells. The plates were incubated at 37° C. for 45minutes. A 10 μl sample was asceptically removed from each well andspread on a BHI-XV plate. The plates were incubated overnight at 37° C.The bactericidal titer was determined as the reciprocal of the highestdilution of serum capable of reducing the number of CFUs resuspended inPCMA at 10 times original volume. The sample was restored to originalvolume using a Centricon microconcentrator unit with a 10,000 molecularweight cut off membrane. The sample was washed in PCM an additional fourtimes as described above to remove residual ammonium sulfate. Polyclonalrabbit sera was not pretreated for use in the BC assay.

Fifteen microliters of the serum sample were placed in the first well ofa sterile 96 well U-bottom microtiter plate held on ice. Two-fold serialdilutions using PCMA were done in the remaining wells. The plates wereremoved from the ice and 15 μl of the cell/complement mixture were addedto the serum in the wells. The plates were incubated at 37° C. for 45minutes. A 10 μl sample was asceptically removed from each well andspread on a BHI-XV plate. The plates were incubated overnight at 37° C.The bactericidal titer was determined as the reciprocal of the highestdilution of serum capable of reducing the number of CFUs by 50% comparedto a nonantibody containing control well.

Results from one such experiment are shown below;

                  TABLE 3    ______________________________________    Bactericidal Activity of Rabbit    Anti-protein "e" Antiserum Against    Non-Typable H. Influenzae Clinical    Isolates                          Anti-protein "e"    Time          Strain  Titer*    ______________________________________    WEEK 0        N90     5    WEEK 6        N90     160    WEEK 0        S2      10    WEEK 6        S2      >640    WEEK 0        0246E   5    WEEK 6        0246E   40    WEEK 0        HST34   5    WEEK 6        HST34   20    ______________________________________     *Reciprical highest dilution capable of killing 50% or more of the NTHi i     the assay.

As can be seen from Table 3, anti-protein "e" antibody has BC activityagainst non-typable H. influenzae strains.

The H. influenzae OMP designated P4, a protein of about 28,000 daltonsmolecular weight, has been shown to be non-protective in the passiveprotection assay in infant rats (Granoff and Munsen, J. Infect. Dis.1986. 153:448-461). All known immunogens of H. influenzae that areprotective in a passive transfer of antibody assay also elicitsbactericidal antibodies. Here we show that the H. influenzae protein "e"of about 28,000 daltons to elicit bactericidal antibodies and thus theseantibodies would be expected to be protective.

Synergy of Anti-protein "e" with Other Antibodies

Other investigators have reported that antibodies against some OMPs canblock the bactericidal activity of antibodies directed against otherOMPs. K. A. Joiner, et al. (1985) J. Clin. Invest. 76:1765-1772. Anassay for BC was performed in. Order to determine whether anti-protein"e" antibodies have blocking effects when antibodies directed againstother Hi components are present. The details of the assay forbactericidal activity of antibody are given above. Bactericidal titersare read as the reciprocal of the highest dilution of an antiseracapable of killing ^(>) 50% of a defined number of bacteria. The assaymay be performed with either nontypable H. influenzae (NTHi) or type bH. influenzae. NTHi show greater serum sensitivity and are thus somewhateasier to kill in the assay, but are more difficult to use. Thebactericidal titers are usually shown with the preimmune and immunesera. This is because of the extremely variable sensitivity of NTHi tokilling by anti-OMP antibodies. Thus titers may cover a wide range.Showing pre and post immune sera allows us to show the specificity ofthe killing no matter what the titer.

To determine if anti-protein "e"-antibody has this blocking effect withantibodies against another OMP, we examined the effect of mixing theanti-protein "e" with antibody against a recombinant Haemophilus outermembrane protein, rPCP. The bactericidal titers of the individualantisera and the mixtures tested are shown in table 4. No blockingeffects were observed. In contrast, the BC titer of the mixture wasalways greater than the titer of either of the individual antisera. Ifthere was no additive effect, one would expect that the BC titer of themixture would be the same as the titer of the more active of theindividual antisera. If there was an additive effect, then the titer ofthe mixture would be expected to be the sum of the titers of theindividual antisera. However, the titers of the mixture show synergywhere the BC titer of the mixture is greater than the sum of the titersof the individual antisera.

                  TABLE 4    ______________________________________    anti-e         anti-rPCP    anti-e & anti-rPCP           Pre-            Pre-         Pre-    Strain immune  Immune  immune Immune                                        immune Immune    ______________________________________    Hst 33 <1/5    1/10     1/10  1/20  1/5    >1/32    N0264E <1/5    1/40    <1/5   1/5   1/5     1/160    N0133E  1/10   1/40     1/10  1/80  1/5     1/160    N1955   1/5    1/40    <1/5   1/20  1/5    >1/640    ______________________________________

IX. Non-Lipidated Form of Protein "e"

In order to create a non-lipidated version of protein "e", site directedmutagenesis was employed. In the amino terminal end of the "e" sequence,a BamHI site was created by site directed mutagenesis using the dut-ungsystem supplied by Biorad Laboratories (Richmond, Calif.). The followingchanges were made in the "e" gene:

Sequence of gene encoding the amino terminus of the mature "e" protein

       ...... | TGT GGT TCA CAC .....    Sig. seq. | Cys Gly Ser His

Changed to

                       BamHI            ...... TGT GGA TCC CAC .....                   Cys Gly Ser His

This was done by cloning the 997 bp EcoRI-DraI fragment containing the"e" gene into the EcoRI-SmaI sites of M13mp19. Single stranded DNA wasisolated after infection of dut,ung E. coli strain CJ236. This DNAcontains uracil residues which replace thymidine residues and isnon-infectious for normal E. coli. The single stranded U-DNA was mixedwith a single stranded primer containing the desired mutations andhomologous flanking sequences and the DNAs annealed slowly. The primerwas extended on the DNA using all four dNTPs and the Klenow fragment ofE. coli polymerase to complete the circle. Wild type E. coli wereinfected with the M13 DNA causing only the newly synthesized strand tobe replicated and inserting the mutation in the gene. The M13 DNAcontaining the "e" gene with the BamHI site was isolated, the geneisolated by digestion with BamHI (a BamHI site also exists 3' to thegene from the multiple cloning site region of the M13mp19) and subdlonedinto pUC8. The resulting clones, designated pPX524, were screened withmonoclonal antibodies to protein "e". After screening with monoclonalantibodies to protein "e" for expressing clones, no positive isolateswere obtained. Analysis of clones showed that all contained the "e"gene, in the reverse (non-expressing) orientation. The signaless "e"gene has been expressed under control of a regulated promotor; toexpress the gene under lac control, the "e" gene was removed from pPX524by digestion with HincII and EcoRI and directionally cloned into pUC9 atthe EcoRI and SmaI sites yielding a fusion with the following jointsequence in plasmid pPX525:

    ATG ACC ATG ATT ACG CCA AGC TTG GCT GCA GGT CGA CGG    met thr met ile thr pro ser leu ala ala gly arg arg    <----------------lac a peptide-------------------->              ATC CCC|GAC GGA TCC CAC CAA              ile pro|asp gly ser his gln    ----"e" gene---->

The fusion is weakly expressed and has been visualized by reactivity tomonoclonal antibodies.

The signaless "e" gene has also been fused onto the PCP-PAL fusion byisolating the HincII fragment containing the signaless "e" gene frompPX525 and ligating it into StuI-EcoRI digested pPX521 which containesthe PCP-PAL fusion. The fusion joint formed is as follows:

    TAC GTA GAG GGA CGG ATC CCC|GAC GGA TCC CAC CAA....    tyr val glu ala arg ile pro|asp gly ser his gln    ----"e" gene----->rotein--> <

Expression of the triple fusion was confirmed by Western blot ofDH5α(F'lacIq) cells containing the triple fusion plasmid with monoclonalantibodies directed against PCP, PAL, and "e" proteins.

Sequences of the fusion joints and the site directed mutagenesis havebeen confirmed by DNA sequencing.

Deposit of Microorganism

E. coli strain JM103 (pPX513) was deposited with the AgriculturalResearch Culture Collection (NRRL) 1815 North University Street, Peoria,Ill. 61604. and has been assigned the accession number NRRL B-18444deposited on Jan. 26, 1989.

E. coli strain bH5α(F'lacIq, pPX525) was deposited with the NRRL on Mar.8, 1990, and has been assigned Accession Number NRRL B-18629.

Equivalents

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

We claim:
 1. Protein "e" of Haemophilus influenzae which issubstantially free of endotoxin contamination, wherein said protein whenadministered to a mammal is capable of raising antibodies in said mammalwhich are protective in the infant rat passive immunization model.
 2. Apreparation of protein "e" of Haemophilus influenzae, wherein saidprotein (a) is substantially free of endotoxin contamination, and (b)when administered to a mammal is capable of raising antibodies in saidmammal which are protective in the infant rat passive immunizationmodel.
 3. Protein "e" of Haemophilus influenzae having the amino acidsequence shown in FIG. 7, wherein said protein (a) is substantially freeof endotoxin contamination, and (b) when administered to a mammal iscapable of raising antibodies in said mammal which are protective in theinfant rat passive immunization model.
 4. Recombinant protein "e" ofHaemophilus influenzae, wherein said protein (a) is substantially freeof endotoxin contamination, and (b) when administered to a mammal iscapable of raising antibodies in said mammal which are protective in theinfant rat passive immunization model.
 5. Protein "e" of claim 4, whichis unlipidated.
 6. Protein "e" of Haemophilus influenzae which issubstantially free of endotoxin contamination, wherein said protein whenadministered to a mammal is capable of raising antibodies in said mammalwhich are bactericidal against nontypable Haemophilus influenzae.
 7. Apreparation of protein "e" of Haemophilus influenzae, wherein saidprotein (a) is substantially free of endotoxin contamination, and (b)when administered to a mammal is capable of raising antibodies in saidmammal which are bactericidal against nontypable Haemolhilus influenzae.8. Protein "e" of Haemophilus influenzae having the amino acid sequenceshown in FIG. 7, wherein said protein (a) is substantially free ofendotoxin contamination, and (b) when administered to a mammal iscapable of raising antibodies in said mammal which are bactericidalagainst nontypable Haemophilus influenzae.
 9. Recombinant protein "e" ofHaemophilus influenzae, wherein said protein (a) is substantially freeof endotoxin contamination, and (b) when administered to a mammal iscapable of raising antibodies in said mammal which are bactericidalagainst nontypable Haemolhilus influenzae.
 10. Protein "e" of claim 9,which is unlipidated.